Cytokine production inhibitors

ABSTRACT

A 5,6-dihydro-α-pyrone of formula (I)                    
     wherein R is CO 2 H or CH 3  and, when R is CO 2 H, a pharmaceutically or veterinarily acceptable salt thereof. Processes for producing compounds of formula (I) and their use as cytokine production inhibitors.

The present invention relates to 5,6-dihydro-α-pyrones useful ascytokine production inhibitors, to the preparation of these compoundsand to pharmaceutical and veterinary compositions containing them.

We have now discovered that fermentation of a strain of the fungusPhomopsis sp. in a nutrient medium produces two 5,6-dihydro-α-pyronesesterified in the 5-position with an unsaturated C₁₄ fatty acid and alsothe free C₁₄ fatty acid. We have also discovered that fermentation of astrain of the fungus Paecilomyces sp. in a nutrient medium produces auseful phomalactone.

The present invention therefore provides a 5,6-dihydro-α-pyrone offormula (I):

wherein R is CO₂H or CH₃ and each of R¹ and R² is H; or R is CO₂H, oneof R¹ and R² is H and the other is OH; or, when R is CO₂H, apharmaceutically or veterinarily acceptable salt thereof.

Preferred compounds of the invention are:

3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-enoicacid; and

3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-ene.

The present invention provides a process for the preparation of a5,6-dihydro-α-pyrone of formula (I) or a pharmaceutically orveterinarily acceptable salt thereof, which process comprises:

(i) fermenting, in a source of carbon, nitrogen and inorganic salts,fungal strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof whichproduces a said 5,6-dihydro-α-pyrone;

(ii) isolating a said 5,6-dihydro-α-pyrone from the fermentation broth;and

(iii) if desired, when the isolated 5,6-dihydro-α-pyrone is the compoundof formula (I) wherein R is CO₂H, converting the said5,6-dihydro-α-pyrone into a pharmaceutically or veterinarily acceptablesalt thereof.

The compounds of formula (I) have been isolated from a microorganismwhich we have designated X22502 and which has been identified as astrain of the genus Phomopsis (Saccardo) Bubák on the basis of thefollowing morphological data with reference to the description given bySUTTON, B. C., 1980 (The Coelomycetes. Farnham Royal: CommonwealthAgricultural Bureaux):

The fungal strain Phomopsis sp. (X22502) (CBS 313.96) is a coelomyceteisolated from tropical freshwater foam which produces a dense, darkgrey-olivaceous (Flora of British Fungi Colour Identification Chart,1969, Edinburgh: HMSO) mycelium with a white lobate margin at 24° C. on2% malt extract agar with glucose and peptone (MEA: composition perliter of distilled water: Difco malt extract, 20 g; Bacto-peptone, 1 g;agar, 20 g). After 7 days the mycelium attains a diameter of 2.5-3.5 cm.

Conidiomatal development is stimulated by exposure to near-UV light.Conidiomata are solitary, carbonaceous, unilocular, ostiolate andmeasure 1.5-2.0 mm wide and 1.25-2.0 mm high. Conidiogenous cells areborne on branched conidiophores which line the conidiogenous cavity.These cells are hyaline, obclavate to cylindrical, integrated, phialidicand measure 16-20 μm×1.5-2.0 μm. Conidia are hyaline, aseptate andgenerally of three types: A-conidia (5.5-7.5 μm×1.5-3.0 μm) areellipsoid to fusiform, usually with acute apices and a guttule at eachend; B-conidia (20-32 μm×<1 μm) are hamate and filiform; C-conidia(9.5-11.5 μm×1.5-3.0 μm) are obclavate with acute apices and usually atleast three guttules. All three conidial types can be found within asingle conidioma. The fluvial origin and observed microscopicalcharacters did not allow further classification to species.

The 5,6-dihydro-α-pyrones of formula (I) are associated primarily withthe mycelium on termination of the fermentation. They may be recoveredand purified from the medium. The separation and purification of thecompounds from the fermentation broth and their recovery can be achievedusing solvent extraction followed by chromatographic fractionation. The5,6-dihydro-α-pyrone of formula (I) in which R is CO₂H may be convertedinto pharmaceutically or veterinarily acceptable salts by conventionalmethods. Suitable salts include salts with alkali metals such as sodiumor potassium and ammonium salts.

The 5,6-dihydro-α-pyrone of formula (I) wherein R is CH₃ can,alternatively, be produced by the esterification of the phomalactone offormula (II):

with (6S)-4,6-dimethyldodeca-2E,4E-dienoic acid which is a fatty acid offormula (IIIa):

Preferably the reaction is carried out in the presence of a dehydratingagent such as DCC (dicyclohexylcarbodiimide) or EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) anddimethylaminopyridine. The reaction is typically carried out in an inertsolvent such as dichloromethane or tetrahydrofuran.

The reagents are generally mixed with stirring for example at a lowtemperature such as −78° C. The reaction is then allowed to warm to roomtemperature (20-25° C.) and stirred until complete. The reaction may bemonitored chromatographically by thin layer chromatography or reversedphase high performance liquid chromatography and is typically completewithin sixteen hours. Other dehydrating agents such as analkylchloroformate and triethylamine, phenyldichlorophosphate,2-chloro-1,3,5-trinitrobenzene and pyridine, and chlorosulphonylisocyanate can also be used under similar conditions.

Alternatively an excess of the phomalactone of formula (II) can bereacted with the acid of formula (IIIa). The water formed can be removedby azeotropic distillation. Suitable solvents include toluene and1,4-dioxane. The reaction is typically catalysed by acids such assulphuric acid and ptoluenesulphonic acid.

It is advantageous to produce the 5,6-dihydro-α-pyrone of formula (I)wherein R is CH₃ by this route as both the phomalactone and the fattyacid can be produced by fermentation in larger quantities than the5,6-dihydro-α-pyrone of formula (I) wherein R is CH₃.

The fatty acid of formula (IIIa) is one of a group of fatty acids of thefollowing formula (III):

wherein one of R¹ and R² is H and the other is H or OH. These fattyacids can be obtained by fermentation of the fungal strain Phomopsis sp22502 (CBS 313.96) or a mutant thereof. In accordance with the presentinvention, therefore, the fatty acid of formula (III) can be produced bya process which comprises:

(i) fermenting, in a source of carbon, nitrogen and inorganic salts,strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof whichproduces the said fatty acid; and

(ii) isolating the said fatty acid of formula (III) from thefermentation broth.

The fatty acid of formula (III) may, of course, be isolated from thesame fermentation broth as the 5,6-dihydro-α-pyrones of formula (I). Thefatty acid, like the 5,6-dihydro-α-pyrones, is primarily associated withthe mycelium on termination of fermentation.

Some of the fatty acids of formula (III) are novel. The presentinvention therefore further provides a fatty acid of formula (IIIb):

wherein one of R¹ and R² is H and the other is OH

The phomalactone of formula (II) can be synthesised by methods known inthe prior art, for example those disclosed in Krivobok, S. et al,Pharmazie, (1994): 49, H8, 605-607; Guirand, P. et al, Pharmazie,(1994): 49, H8, 279-281; Krasnoff, S. B. et al, J. Chem. Ecol., (1994):20, 293-302 and Murayama, T. et al, Agric. Biol. Chem., (1987): 51,2055-2060.

The present invention does however provide a new process for thepreparation of the phomalactone of formula (II), which processcomprises:

(i) fermenting, in a source of carbon, nitrogen and inorganic salts,fungal strain Paecilomyces sp. 3527 (CBS 314.96) or a mutant thereofwhich produces the said phomalactone; and

(ii) isolating the said phomalactone from the fermentation broth.

The phomalactone is found primarily in the culture liquor on terminationof the fermentation and may be recovered and purified. The separationand purification of the compound from the fermentation broth and itsrecovery can be achieved using solvent extraction followed byapplication of conventional chromatographic fractionations with variouschromatographic techniques and solvent systems.

The phomalactone of formula (II) has been isolated from a microorganismwhich we have designated X3527 and which has been identified as a strainof the genus Paecilomyces Bainier on the basis of the followingmorphological data with reference to the descriptions given by SAMSON,R. A., 1974 (Paecilomyces and some allied Hyphomycetes. Studies inMycology No. 6. Baarn: CBS). The fungal strain Paecilomyes sp. (X3527)(CBS 314.96) is an entomogenous hyphomycete isolated from a tropicalLepidoptera pupa which produces white mycelium attaining 4.5-5.0 cmdiameter within 14 days at 25° C. on 2% MEA. The aerial mycelium becomespowdery as conidiogenesis occurs and may develop denser concentriczones. A pale-buff-yellow pigmentation frequently develops in aerialand/or submerged mycelium.

Conidiophores are hyaline and smooth-walled with stipe dimensions of100-400 μm×1.5-2.5 μm and bear single or sparsely clustered phialides.Phialides are produced with the characteristic morphology ofPaecilomyces Bainier measuring 7-20 μm long with a lower sectioninflated to 2-2.5 μm wide. The phialide neck is often considerablyattenuated (<1 μm wide) and bent. Phialides of very variable morphologyare also produced, e.g. lacking an inflated basal region and/or with anattenuated neck measuring about 20 μm long. Conidia are ellipsoid tocylindrical (3.5-7 μm×2-3 μm), smooth-walled, hyaline and borne inconspicuous imbricate dry chains.

Further classification as a Paecilomyces anamorph of Cordyceps (Fries)Link may be justified on the grounds of the epidopterous origin of theisolate coupled with the results of numerous studies made by H. C. Evansand R. A. Samson (unpublished data) of entomopathogenic Paecilomycesanamorphs of Cordyceps.

The strains X22502 and X3527 were deposited by Xenova Group plc of 240Bath Road, Slough, Berkshire, SL1 4EF, United Kingdom under the BudapestTreaty at the Centraalbureau voor Schimmelcultures, Baarn, theNetherlands, on 19th Mar. 1996 under references X07/64/502 andX08/64/527 respectively. Strain X22502 was assigned the reference numberCBS 313.96. Strain X3527 was assigned the reference number CBS 314.96.

The present invention also embraces mutants of the above microorganisms.For example, those which are obtained by natural selection or thoseproduced by mutating agents including ionising radiation such asultraviolet irradiation, or chemical mutagens such as nitrosoguanidineor the like treatments, are also included within the ambit of thisinvention.

The invention further provides a biologically pure culture of fungalstrain X22502 or X3527 or of a mutant thereof which produces thecompounds of the invention. Such cultures are substantially free fromother microorganisms. The invention also provides a process forfermenting the fungal strain X22502, X3527 or a said mutant, whichprocess comprises fermenting strain X22502 or X3527 or a said mutantthereof in a source of carbon, nitrogen and inorganic salts.

Assimilable sources of carbon, nitrogen and minerals may be provided byeither simple or complex nutrients. Sources of carbon will generallyinclude glucose, maltose, starch, glycerol, molasses, dextrin, lactose,sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols,alkanes and vegetable oils. Sources of carbon will generally comprisefrom 0.5 to 10% by weight of the fermentation medium.

Sources of nitrogen will generally include soya bean meal, corn steepliquors, distillers' solubles, yeast extracts, cottonseed meal,peptones, ground nut meal, malt extract, molasses, casein, amino acidmixtures, ammonia (gas or solution), ammonium salts or nitrates. Ureaand other amides may also be used. Sources of nitrogen will generallycomprise from 0.1 to 10% by weight of the fermentation medium.

Nutrient mineral salts which may be incorporated into the culture mediuminclude the generally used salts capable of yielding sodium, potassium,ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium,chromium, calcium, copper, molybdenum, boron, phosphate, sulphate,chloride and carbonate ions.

An antifoam may be present to control excessive foaming and added atintervals as required.

Fermentation can be conducted at temperatures ranging from 20° C. to 40°C., preferably 24-30° C. For optimal results, it is most convenient toconduct these fermentations at a temperature in the range 24-26° C. Thestarting pH of the nutrient medium suitable for producing the compoundscan vary from 5.0 to 8.5 with a preferred range of from 5.0 to 7.5.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask by known steriletechniques, inoculating the flask with either spores or vegetativecellular growth of the fungal strain, loosely stoppering the flask withcotton wool, and permitting the fermentation to proceed in a constantroom temperature of about 25° C. on a rotary shaker at from 95 to 300rpm for 2 to 10 days. The fermentation may also be conducted in staticculture on liquid or semi-solid medium.

For larger scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. The nutrient medium is made up in the tank aftersterilization and is inoculated with a source of vegetative cellulargrowth of the fungal strain. The fermentation is allowed to continue forfrom 1 to 8 days while agitating and/or aerating the nutrient medium ata temperature in the range 20° C. to 37° C. The degree of aeration isdependent upon several factors such as the size of the fermenter andagitation speed. Generally the larger scale fermentations are agitatedat about 95 to 750 rpm and aerations of about 0.5 to 1.5 VVM (volumes ofair per volume of medium per minute).

The separation of the present compounds from the whole fermentationbroth and their recovery is carried out by solvent extraction followedby application of chromatographic fractionations with variouschromatographic techniques and solvent systems. The present compounds inpure form have thus been isolated in this way.

The 5,6-dihydro-α-pyrones of formula (I) and pharmaceutically andveterinarily acceptable salts of the compound of formula (I) wherein Ris CO₂H are inhibitors of the production of cytokines, specificallyIL-1β.

These compounds can therefore be used in the treatment of disordersrequiring immunosuppression, for example immunoinflammatory conditionsand CNS disorders. A human or animal, e.g. a mammal, can therefore betreated by a method comprising administration of a therapeuticallyeffective mount of a compound of formula (I), or a pharmaceutically orveterinarily acceptable salt of the compound of formula (I) wherein R isCO₂H.

These compounds can be used in the treatment of an immunoinflammatorycondition such as rheumatoid arthritis, osteoarthritis, septic shock,psoriasis, atherosclerosis, inflammatory bowel disease, Crohn's diseaseand asthma. The compounds of the present invention also exhibitpharmacological properties associated with the treatment of otherdisorders requiring immunosuppression, for example central nervoussystem (CNS) disorders such as encephalomyelitis and Alzheimer'sdisease.

The compounds of the present invention can be administered in a varietyof dosage forms, for example orally such as in the form of tablets,capsules, sugar- or film-coated tablets, liquid solutions or suspensionsor parenterally, for example intramuscularly, intravenously orsubcutaneously. The present compounds may therefore be given byinjection or infusion.

The dosage depends on a variety of factors including the age, weight andcondition of the patient and the route of administration. Typically,however, the dosage adopted for each route of administration for adulthumans is 0.001 to 10 mg/kg, most commonly in the range of 0.01 to 5mg/kg, body weight. Such a dosage may be given, for example, from 1 to 5times daily orally or by bolus infusion, infusion over several hoursand/or repeated administration.

The toxicity of the compounds of the invention is negligible, they cantherefore safely be used in therapy.

The compounds of the present invention are formulated for use as apharmaceutical or veterinary composition also comprising apharmaceutically or veterinarily acceptable carrier or diluent. Thecompositions are typically prepared following conventional methods andare administered in a pharmaceutically or veterinarily suitable form.

For example, the solid oral forms may contain, together with the activecompound, diluents, such as lactose, dextrose, saccharose, cellulose,corn starch or potato starch; lubricants such as silica, talc, stearicacid, magnesium or calcium stearate and/or polyethylene glycols; bindingagents such as starches, arabic gums, gelatin, methylcellulose,carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agentssuch as starch, alginic acid, alginates or sodium starch glycolate;effervescing mixtures; dye-stuffs; sweeteners; wetting agents such aslecithin, polysorbates, laurylsulphates. Such preparations may bemanufactured in known manner, for example by means of mixing,granulating, tabletting, sugar coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions andsuspensions. The syrups may contain as carrier, for example, saccharoseor saccharose with glycerol and/or mannitol and/or sorbitol. Inparticular a syrup for diabetic patients can contain as carriers onlyproducts, for example sorbitol, which do not metabolise to glucose orwhich only metabolise a very small amount to glucose. The suspensionsand the emulsion may contain as carrier, for example a natural gum,agar, sodium alginate, pectin, methylcellulose, carboxymethylcelluloseor polyvinyl alcohol.

Suspensions or solutions for intramuscular injections may contain,together with the active compound, a pharmaceutically acceptable carriersuch as sterile water, olive oil, ethyl oleate, glycols such aspropylene glycol, and if desired, a suitable amount of lidocainehydrochloride. Solutions for intravenous injection or infusion maycontain a carrier, for example, sterile water which is generally Waterfor Injection. Preferably, however, they may take the form of a sterile,aqueous, isotonic saline solution. Alternatively, the compounds of thepresent invention may be encapsulated within liposomes.

The following examples illustrate the invention

EXAMPLE 1

Batch Fermentation of Phomopsis sp. 22502

A 1.5 ml cryovial containing 1 ml of macerated vegetative myceliumsuspended in a 10% glycerol solution was retrieved from storage at −135°C. A preculture was produced by aseptically placing 1 ml of startingmaterial in a 250 ml baffled Erlenmeyer flask containing 40 ml ofnutrient solution S1 and shaken at 240 rpm for 3 days at 25° C.

An intermediate culture was generated by aseptically transferring thepreculture to 2 L of nutrient solution S1 in a 3 L fermenter. Thefermenter was agitated at 500 rpm, aerated at 0.5 vvm, and thetemperature controlled at 25° C. for 3 days.

A production culture was generated by aseptically transferring anintermediate culture to a 75 L fermenter containing 50 L of nutrientsolution P1. The production fermenter was stirred at 350 rpm, aerated at0.5 vvm, and temperature controlled at 25° C. After 5½ days incubationthe fermentation was stopped and the culture was harvested.

The nutrient solutions used were as follows, percentages being byweight:

Nutrient Solution S1: 1.5% glycerol, 1.5% soya bean peptone, 1%D-glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO₃, 0.1%: Tween 80,0.1% Junlon PW110 [suppliers: Honeywell and Stein, Sutton, Surrey, U.K.]adjusted to pH 6

Nutrient Solution P1: 3.6% molasses, 0.2% casein hydrolysate, 0.004%phytic acid, 0.09% calcium chloride, 0.1%: Tween 80, adjusted to pH 5

EXAMPLE 2

Extraction and purification of the 5,6-dihydro-α-pyrones of formula (I)wherein R¹ and R² are both H, and the fatty acid of formula (IIIa) fromPhomopsis sp. 22502

The broth in Example 1 was harvested by filtration using a Schenk Niro430 filter press, the clarified filtrate was discarded and the retainedbiomass was extracted with 25 L of recirculating methanol for 24 hours.The methanolic extract was harvested via filtration through the filterpress and evaporated to an aqueous concentrate using a thin filmevaporator.

The aqueous concentrate (10 L) was then back extracted with 2×7 L of anethyl acetate:hexane (1:1) mix. The solvent extracts were pooled andevaporated to a gum under reduced pressure and redissolved in 50 ml ofethyl acetate:hexane (1:1). Purification was achieved by normal phasechromatography using a Biotage Flash 75 chromatography system and aFlash 75 KP-Sil silica (32-62 μm 60 Å) column (internal diameter (ID)7.5×30 cm length) and an isocratic mobile phase (ethyl acetate:hexane1:1 mix, 200 ml/min flow rate). 1 L fractions were collected andanalysed by thin layer chromatography using the same mobile phase as thedeveloping solvent.

The 5,6-dihydro-α-pyrone of formula (I) wherein R is CO₂H (Rf 0.53), the5,6-dihydro-α-pyrone of formula (I) wherein R is CH₃ (Rf 0.89) and thefatty acid of formula (IIIa) (Rf 0.75) rich fractions were pooled,evaporated to dryness under reduced pressure and subjected to furtherpurification by preparative reversed phase HPLC using a Waters NovaPakC18 (100 Å 5 μM) column (ID 2.5×20 cm length) and an isocratic mobilephase (80% acetonitrile: 20% water plus 0.1% v/v glacial acetic acid,flow rate 50 ml/min). Wavelength monitoring was at 278 nm. The peakscollected at 11-14 minutes, 19-21 minutes and 30-32 minutes wereevaporated to dryness to yield the 5,6-dihydro-α-pyrone of formula (I)wherein R is CO₂H (7.5 g), the fatty acid of formula (IIIa) (0.35 g) andthe 5,6-dihydro-α-pyrone of formula (I) wherein R is CH₃ (0.9 g),respectively.

Physicochemical data for the three compounds are set out in Tables 1 to3 below. Tables 2 and 3 show ¹H and ¹³C NMR assignments respectively.

TABLE 14 5,6-dihydro-α- fatty Acid of pyrone of formula (I) formula R isCO₂H R is CH₃ (IIIa) DCI-MS (m/z) 391 (MH)⁺ 361 (MH)⁺ 225 (NH)⁺Molecular C₂₂H₃₀O₆ C₂₂H₃₂O₄ C₁₄H₂₄O₂ formula UVλ_(max)nm 206, 276 204,274 265 IR (KBr)ucm⁻¹ 3391, 2960, 2928, 3390, 2960, 2929, 2900, 2685,2855, 1717, 1620, 1720, 1615, 1560, 2589 1570, 1396, 1285, 1250, 1180,1010, 1687, 1618, 1250, 1161, 1024, 980. 1459 980. 1417, 1285, 12071028, 984, 940, 952, 700

TABLE 2 †δH/ppm in MeOH-d4 5,6-dihydro-α-pyrone of formula (I) FattyAcid of Position R is CO₂H R is CH₃ formula (IIIa)*  2  3 6.35 (1H, d,9.7) 6.29 (1H, d, 9.8)  4 7.21 (1H, dd, 9.7, 7.18 (1H, dd, 9.8, 5.6)5.6)  5 5.64 (1H, dd, 5.6, 5.48 (1H, dd, 5.6, 3.1) 3.0)  6 5.49 (1H, m)5.19 (1H, ddq, 7.2, 3.0, 0.9)  7 6.91 (1H, dd, 5.72 (1H, ddq, 15.4,15.7, 5.0) 7.2, 1.7)  8 6.32 (1H, dd, 6.05 (1H, dqd, 15.4, 15.7, 1.8)6.6, 1.1)  9 1.84 (3H, ddd, 6.8, 1.7, 0.8) 1′ 1′-OH 11.70 (1H, br s)  2′5.88 (1H, d, 15.6) 5.95 (1H, dd, 15.7, 5.77 (1H, d, 0.6) 15.6)  3′ 7.41(1H, dd, 7.41 (1H, dd, 15.6, 7.39 (1H, d, 15.6, 0.6) 0.8) 15.6)  4′  5′5.83 (1H, br d, 5.83 (1H, br d, 9.2) 5.72 (1H, d, 9.8) 9.8)  6′ 2.66(1H, m) 2.65 (1H, m) 2.53 (1H, m)  7′-11′ 1.3-1.5 (10H, m) 1.4 (10H, m)1.2-1.4 (10H, m) 12′ 0.97 (3H, t, 6.9) 0.95 (3H, t, 6.9) 0.87 (3H, t,6.7) 13′ 1.87 (3H, d, 1.1) 1.81 (3H, d, 1.2) 1.78 (3H, d, 0.8) 14′ 1.08(3H, d, 6.6) 1.09 (3H, d, 6.6) 0.98 (3H, d, 6.6) *values obtained inCDCl₂ †The J values are in parenthesis (Hz)

TABLE 3 δC/ppm in MeOH-d4 5,6-dihydro-α-pyrone Fatty Acid of formula (I)of formula Position R is CO₂H R is CH₃ (IIIa)  2 164.6 165.5  3 125.6126.1  4 143.4 143.4  5 64.9 65.6  6 79.6 81.4  7 139.6 125.5  8 128.2138.8  9 170.7 18.2  1′ 168.0 168.0 173.1  2′ 115.3 115.5 114.7  3′153.5 153.2 152.1  4′ 133.3 133.1 131.2  5′ 151.7 151.5 149.8  6′ 35.034.8 33.2  7′ 38.7 36.6 37.1  8′ 33.4 33.2 31.8  9′ 30.9 30.7 29.3 10′29.0 28.9 27.3 11′ 24.1 23.9 22.5 12′ 14.8 14.6 13.9 13′ 12.9 12.7 12.114′ 21.1 20.9 20.2 *values obtained in CDCl₂

EXAMPLE 3

Extraction and purification of the 5,6-dihydro-α-pyrones of formula (I)wherein one of R¹ and R² is H and the other is OH, and the fatty acidsof formula (IIIb) from Phomopsis sp 22502.

The title compounds all possess a hydroxy substituent (as R¹ or R²) andare therefore more polar analogues of the compounds of formulae (I) and(IIIa) produced as described in Example 2.

The title compounds were isolated from the broth of Example 1 as minorfermentation components, using purification methods similar to thosedescribed in Example 2.

Physicochemical data for the three compounds are set out in thefollowing Tables 4 to 6. Tables 5 and 6 show ¹H and ¹³C NMR assignments,respectively.

TABLE 4 Compound of formula (I) Fatty acid of formula (IIIb) A B C D (R¹= H, (R¹ = OH, (R¹ = H, (R¹ = OH, R² = OH) R² = H) R² = OH) R² = H)DCI-MS (m/Z) 424 424 258 258 (MNH₄+) (MNH₄+) (MNH₄+) (MNH₄+) 407 (MH+)407 (MH+) 240 (MH+) 240 (MH+) Molecular C₂₂H₁₀O₇ C₂₂H₃₀O₇ C₁₄H₂₄O₃C₁₄H₂₄O₃ formula UV λ_(max) nm 203, 275 205, 274 267 266 IR(KBr)u 3444,2956, 3400, 2900, 3255, 2900, 3363, 1692, cm⁻³ 1723, 1621, 1714, 1620,1694, 1627, 1622, 1285, 1289, 1249, 1286, 1247, 1382, 1285 1198, 1029,1159, 1107, 1156, 1106, 1193, 982 983 981 980

TABLE 5 Posi- δH/ppm in MeOH-d4 tion A B C D  2  3 6.35 (1H, d, 9.8)6.35 (1H, d, 9.8)  4 7.21 (1H, dd, 9.7, 7.21 (1H, dd, 5.7) 9.8, 5.8)  55.67 (1H, dd, 5.7, 5.67 (1H, dd, 3.0) 6.3, 5.3)  6 5.52 (1H, m) 5.52(1H, m)  7 6.99 (1H, dd, 6.98 (1H, dd, 15.7, 4.5) 15.7, 4.7)  8 6.30(1H, dd, 6.31 (1H, dd, 15.7, 1.8) 15.7, 1.9)  9  1′  2′ 5.87 (1H, d,15.5) 5.87 (1H, d, 5.87 (1H, d, 5.85 (1H, d, 15.6) (15.6) 15.6)  3′ 7.40(1H, d, 15.3) 7.40 (1H, d, 7.38 (1H, d, 7.40 (1H, d, 15.5 15.9) 15.7) 4′  5′ 5.84 (1H, brd, 5.83 (1H, brd, 5.75 (1H, brd, 5.75 (1H, d, 9.0)9.9) 9.8) 9.8)  6′ 2.67 (1H, m) 2.67 (1H, m) 2.68 (1H, m) 2.65 (1H, m) 7′ 1.3-1.5 (8H, m) 1.3-1.55 1.35-1.6 1.4-1.6 (8H, m) (8H, m) (8H, m) 8′ ″ 1.3-1.55 1.35-1.6 1.4-1.6 (8H, m) (8H, m) (8H, m)  9′ 3.50 (1H, m)1.3-1.55 3.52 (1H, m 1.4-1.6 (8H, m) (8H, m) 10′ 1.3-1.5 (8H, m)1.3-1.55 1.35-1.6 1.4-1.6 (8H, m) (8H, m) (8H, m) 11′ ″ 3.77 (1H, m)1.35-1.6 3.78 (1H, m) (8H, m) 12′ 1.00 (3H, t, 7.4) 1.23 (3H, d, 1.00(3H, t, 1.20 (3H, d, 6.1) 7.4) 6.2) 13′ 1.87 (3H, s) 1.88 (3h, d, 1.90(3H, d, 1.90 (3H, s) 1.0) 0.8) 14′ 1.09 (3H, d, 6.6) 1.08 (3H, d, 1.10(3H, d, 1.10 (3H, d, 6.6) 6.6) 6.6)

TABLE 6 δC/ppm in MeOH-d4 Position A B C D  2 164.0 164.0  3 125.2 125.2 4 142.8 142.8  5 64.2 64.2  6 78.9 78.9  7 141.2 141.2  8 125.4 125.5 9 168.7 168.8  1′ 167.5 167.6 171.0 171.0  2′ 114.8 114.8 116.8 116.8 3′ 153.2 153.1 151.6 151.6  4′ 132.9 132.9 132.9 132.8  5′ 151.2 151.2149.6 149.7  6′ 34.6 34.6 34.5 34.4  7′ 31.1 38.3 31.1 38.4  8′ 38.428.9 38.5 28.7  9′ 73.9 27.0 73.9 27.0 10′ 38.0 40.2 38.0 40.2 11′ 24.868.6 24.9 68.6 12′ 12.5 23.5 12.6 23.6 13′ 10.3 12.5 10.3 12.6 14′ 20.720.7 20.8 20.9

EXAMPLE 4

Batch Fermentation of Paecilomyces sp. 3527

Starting material of the strain Paecilomyces sp. 3527 was generated bysuspending a mature slant culture, grown on MEA (2% malt extract, 1.5%agar), in 5 ml 10% aqueous glycerol. 1 ml of this suspension, in a 1.5ml cryovial, comprises the starting material which was retrieved fromstorage at −135° C. A preculture was produced by aseptically placing 1ml of starting material in a 250 ml baffled Erlenmeyer flask containing40 ml of nutrient solution S2 shaken at 240 rpm for 3 days at 25° C.

An intermediate culture was generated by aseptically transferring thepreculture to 2 L of nutrient solution S2 in a 3 L fermenter. Thefermenter was agitated at 500 rpm, aerated at 0.5 vvm, and thetemperature controlled at 25° C. for 3 days.

A production culture was generated by aseptically transferring anintermediate culture to a 75 L fermenter containing 50 L of nutrientsolution P2. The production fermenter was stirred at 300 rpm, aerated at0.5 vvm, and temperature controlled at 25° C. During the productionfermentation the pH was uncontrolled and remained between 5.5 and 6.5.In addition the dissolved oxygen tension remained above 80%. After 5days incubation the fermentation was stopped and the culture washarvested.

The solutions used were as follows, percentages being by weight:

Nutrient Solution S2: 1.5% glycerol, 1.5% soya bean peptone, 1%D-glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO₃, 0.1% Tween 80, 0.1%Junlon PW110 (Honeywell and Stein, Sutton, Surrey, U.K.) adjusted to pH6

Nutrient Solution P2: 3.65% sucrose, 1.20% glutamic acid (sodium salt),0.02% K₂HPO₄, 0.98% MES, 0.05% KCl, 0.1% Tween 80, 0.002% MgSO₄, 0.002%CaCl₂, 2% vitamin mix solution (see below), 0.5% trace elements solution(see below), adjusted to pH 6

vitamin mix solution: 0.0025% thiamine, 0.0025% riboflavin, 0.0025%pantothenate, 0.0025% nicotinic acid, 0.0025% pyridoxine, 0.0025%thioctic acid, 0.00025% folic acid, 0.00025% biotin, 0.00025%cyanocobalamin, 0.00025% p-amino benzoic acid, 0.005% vitamin K, 0.2%Tween 80.

trace elements solution: 0.17% ZnSO₄, 0.11% FeSO₄, 0.02% MnSO₄, 0.006%H₃BO₃, 0.012% CuSO₄, 0.005% Na₂MoO₄, 0.005% CoCl₂, 0.008% KI.

EXAMPLE 5

Extraction and Purification of the Phomalactone from Paecilomyces sp.3527

The fermentation broth from Example 4 was harvested by filtration andthe clarified filtrate was divided equally into 3 aliquots and eachaliquot was back extracted batch wise with 10 L of hexane to removenon-polar impurities. The hexane extracts were separated and discarded.Each batch of filtrate was then back extracted with 2×8 L of ethylacetate. The phomalactone and some impurities were extracted into thesolvent while many of the more polar impurities remained in the aqueousphase. The ethyl acetate extracts were pooled and concentrated todryness under reduced pressure and redissolved in 50 ml of ethylacetate.

Purification was achieved by normal phase chromatography using a BiotageFlash 75 chromatography system and a Flash 75 KP-Sil silica (32-62 μm 60Å) column (internal diameter 7.5×30 cm length) and an isocratic mobilephase (100% ethyl acetate, 200 ml/min flow rate). 1 L fractions werecollected and analysed by thin layer chromatography using ethyl acetateas the developing solvent.

Phomalactone rich fractions (Rf 0.75) were pooled, evaporated to drynessunder reduced pressure and subjected to further purification bypreparative reversed phase HPLC using a Beckman 350 HPLC with a ShandonHyper prep HS BOS C18 (100 Å 12 μm) column (internal diameter 10×30 cmlength) and an isocratic mobile phase (85% water:15% acetonitrile, flowrate 170 ml/min). Wavelength monitoring was at 210 nm. The peakcollected between 6-12 minutes was evaporated to dryness to yield thetarget phomalactone (6.0 g).

Physicochemical data for the phomalactone are set out in Tables 7 to 9below. Tables 8 and 9 show ¹H and ¹³C NMR assignments respectively.

TABLE 7 Phomalactone of formula (II) DCI-MS (m/z) 155 (MH)⁺ Molecularformula C₈H₁₀O₃ UVλ_(max)nm 200 IR (KBr)ucm⁻¹ 3425, 2923, 2855, 1716,1629, 1381, 1262, 1157, 1102, 1076, 1034, 969, 830.

TABLE 8 †δH/ppm in CDCl₃ Position Phomalactone of formula (II) 2 3 6.12(1H, d, 9.6) 4 6.97 (1H, dd, 9.7, 5.2) 5 4.21 (1H, dd, 5.2, 3.2) 6 4.85(1H, m) 7 6.00 (1H, dqd, 15.3, 6.5, 1.0) 8 5.75 (1H, ddq, 17.0, 6.9,3.4) 9 1.82 (3H, ddd, 6.1, 1.0) †The J values are in parenthesis (Hz)

TABLE 9 δC/ppm in CDCl₃ Position Phomalactone of formula (II) 2 162.9 3122.8 4 144.3 5 63.1 6 80.9 7 132.9 8 123.8 9 17.8

EXAMPLE 6

Synthesis of the 5,6-dihydro-α-pyrone of formula (I) wherein R¹ and R²are H and R is CH₃ by esterification of the phomalactone with the fattyacid of formula (IIIa).

A solution of the phomalactone (20 mg, 0.13 mmol 1 eq) in drydichloromethane (2 ml) was added to a stirred solution of the fatty acidof formula (III) (29 mg, 0.13 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (24.8 mg,0.13 mmol, 1 eq) and dimethylaminopyridine (1 mg) in dry dichloromethane(5 ml) at −78° C. The reaction mixture was allowed to warm to roomtemperature and was stirred for a further 16 hours. The reaction mixturewas washed with saturated ammonium chloride solution (3×5 ml), water(1×5 ml) and brine (1×5 ml). The organic extract was dried over MgSO₄,filtered and concentrated in vacuo yielding a yellow oil. Purificationby preparative reversed phase HPLC yielded the 5,6-dihydro-α-pyrone offormula (I) wherein R¹ and R² are H and R is CH₃ (11.1 mg). HPLC, TLC,and ¹H NMR analysis showed that this product was identical to theisolated natural product.

EXAMPLE 7

TNP-α Release from U937 Cells

The effect of the compounds of the invention on TNF-α release wasinvestigated using a known method (Lozanski, G. et al., (1992), J.Rheumatol; 19, 921-26).

The human histolytic lymphoma U937 cell line was obtained from acommercial source (ECACC, Salisbury, UK) and maintained in RPMI 1640medium supplemented with 2 mM L-glutamine and 5% fetal bovine serum. Thecells were pretreated with 25 ng/ml PMA for six hours and then exposedto dose ranges of the compound to be tested followed by the addition of1 ng/ml LPS.

After 18 hours incubation at 37° C. with 5% CO₂ the cell culturesupernatants were harvested and stored at −70° C., until required fordetermination of TNF-α secretion by Dissociation Enhanced LanthanideFluorescence Immuno Assay (DELFIA). The effect of the compounds on cellcytotoxicity was measured using the tetrazolium salt, XTT(2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilidesalt) and effects on protein synthesis were determined by investigationof [³H] Leucine uptake.

In this test the compounds of the invention were found to inhibit TNF-αrelease at concentrations of from 10 to 0.1 μM (Table 10). At thisconcentration range, the compounds were not toxic and showed no effecton protein synthesis. The IC₅₀(μM) values for LPS-induced TNF productionin U937 cells for the 5,6-dihydro-α-pyrones for formula (I) are given inTable 11.

EXAMPLE 8

IL-1 Release from Human Monocytes

The effect of the compounds of the invention on IL-Iβ release wasinvestigated using a known method (Bakouche, O. et al., (1992), J.Immunol: 148, 84-91). Human monocytes were purified by elutriation fromBuffy coats obtained from normal healthy donors after the separation ofperipheral blood mononuclear cells (PBMC) on Lymphoprep. The freshlyisolated monocytes were suspended in RPMI 1640 supplemented with 5% FBSand exposed to dose ranges of the compound to be tested followed by theaddition of 1 ng/ml LPS. Cells were incubated for 18 hours at 37° C.with 5% CO₂ and the cell culture supernatants harvested and stored at−70° C. Effects on the production of IL-1β were determined using anELISA.

The compounds of the invention were found to inhibit the release ofIL-1β at concentrations of from 0.2 to 10 μM (Table 12). At thisconcentration range the compounds were not toxic to monocytes. The IC₅₀(μM) values for LPS-induced IL-1β production for the5,6-dihydro-α-pyrones of formula (I) are given in Table 11.

TABLE 10 Inhibition of TNF-α release Concentration μM % Inhibition ofTNF-α R in formula (I) 8 107 is CO₂H 2 100 0.5 77 0.12 47 R in formula(I) 2.8 99 is CH₃ 0.7 90 0.17 61 0.04 20 0.01 0.85

TABLE 11 IC₅₀s for inhibition of LPS-induced cytokine production by the5,6-dihydro-α-pyrones of formula (I) IC₅₀ (μM) R¹, R² in R inLPS-induced TNF LPS-induced IL-β formula Formula production in U937production (I) (I) cells (monocytes) H, H CO₂H 2 2 † H, H CH₃ 0.08 0.19H, OH CO₂H 32 — OH, H CO₂H 31 —

TABLE 12 Inhibition of the release of Il-1β Concentration μM %Inhibition of IL-1β R in formula (I) 20 99 is CO₂H 2 81.5 0.5 14 R informula (I) 5.5 88 is CH₃ 1.1 72 0.22 41 0.044 23

What is claimed is:
 1. A 5,6-dihydro-α-pyrone of formula (I)

wherein R is CO₂H or CH₃ and each of R¹ and R² is H; or R is CO₂H, oneof R¹ and R² is H and the other is OH; or, when R is CO₂H, apharmaceutically or veterinarily acceptable salt thereof.
 2. A processfor the preparation of a 5,6-dihydro-α-pyrone of formula (I) as definedin claim 1 or a pharmaceutically or veterinarily acceptable saltthereof, which process comprises: (i) fermenting, in a source of carbon,nitrogen and inorganic salts, fungal strain Phomopsis sp. 22502 (CBS313.96) or a mutant thereof which produces a said 5,6-dihydro-α-pyrone;(ii) isolating a said 5,6-dihydro-α-pyrone from the fermentation broth;and (iii) if desired when the isolated said 5,6-dihydro-α-pyrone is thecompound of formula (I) wherein R is CO₂H, converting the said5,6-dihydro-α-pyrone into a pharmaceutically or veterinarily acceptablesalt thereof.
 3. A process for the preparation of a 5,6-dihydro-α-pyroneof formula (I), as defined in claim 1, wherein R is CH₃, which processcomprises esterifying the phomalactone of formula (II):

with a fatty acid of formula (IIIa):


4. A pharmaceutical or veterinary composition comprising apharmaceutically or veterinarily acceptable carrier or diluent and, asactive ingredient, a compound as claimed in claim
 1. 5. A method oftreating a patient in need of a cytokine production inhibitor, whichmethod comprises administering thereto a therapeutically effectiveamount of a compound as defined in claim
 1. 6. A method according toclaim 5 wherein the cytokine production inhibitor is an IL-1 productioninhibitor.
 7. A method of treating a clinical condition requiringimmunosuppression, which method comprises administering to a patient inneed thereof a therapeutically effective amount of a compound as definedin claim
 1. 8. A method according to claim 7 wherein said clinicalcondition is an immunoinflammatory condition.
 9. A method according toclaim 8 wherein said immunoinflammatory condition is selected from thegroup consisting of rheumatoid arthritis, osteoarthritis, septic shock,psoriasis, atherosclerosis, inflammatory bowel disease, Crohn's diseaseand asthma.
 10. A method according to claim 7 wherein said clinicalcondition is a central nervous system disorder.
 11. A method accordingto claim 10 wherein said central nervous system disorder is selectedfrom the group consisting of encephalomyelitis and Alzheimer's disease.